Verification device for optical clinical assay systems

ABSTRACT

A device and method for verifying correct performance of an optical clinical assay system is provided.

This application is a continuation of application Ser. No. 10/770,786,filed Feb. 2, 2004, now abandoned, which is a continuation ofapplication Ser. No. 09/564,390, filed May 1, 2000, now abandoned, whichis a continuation of application Ser. No. 09/138,824, filed Aug. 24,1998, now U.S. Pat. No. 6,061,128, which claims the benefit ofProvisional Application No. 60/057,903, filed Sep. 4, 1997.

BACKGROUND OF THE INVENTION

Increasingly in modern clinical chemistry, whole blood samples, oftenobtained by finger stick methods, are analyzed using automated automaticanalysis systems (meters) which employ disposable (often one-time use)test elements, and a non-disposable electronic test device that analyzesthe reaction occurring in the whole blood sample in the disposable testelement, and then outputs an answer. Such systems are used for analyzingwhole blood samples for glucose, cholesterol, and increasingly, morecomplex tests such as coagulation testing (prothrombin time, activatedpartial thromboplastin time), enzymatic analytes, and the like.

Because the answer from these devices are often used to make a clinicaldecision that can significantly impact the health and well-being of apatient, verification methods to insure that the analytical devices areperforming correctly are of obvious importance.

One common method for verifying correct performance of a clinicalanalytical system is through the use of control solution, which isusually a liquid chemical solution with known reactivity. If theanalytical device gives the correct answer with a known referencechemical, then the overall performance of the system can be assessed.

With modern one-shot, disposable test elements, however, the problemwith liquid control testing is that it is destructive. The disposabletest element has been destroyed as a result of the testing, and only thenow-validated meter now survives to test the actual sample. For thisreason, modern verification methods tend to shift validation ofdisposable test elements to manufacturers, who validate batches ofdisposable test elements by statistical sampling methods. The problem ofmeter verification remains, however. Meters are typically used foryears, and can be exposed to environmental extremes, misuse, andmechanical shock.

Because meter verification using liquid control devices and disposabletest units is an expensive process, and because test unit verificationis inherently best suited to statistical lot testing by themanufacturer, there is a need for low-cost methods that can verify theperformance of the meter without the use of liquid control solution anddisposable test units.

Analytical devices for temperature sensitive enzymatic analytes, such asblood coagulation, typically have a temperature controlled reactionstage, means to determine the start of the enzymatic reaction, opticalmeans to access the progress of the reaction, and computational means(typically a microprocessor or microcontroller) to interpret theprogress of the reaction and output an answer. To completely verify theperformance of the analysis system, each subsystem must be assessed. Thetemperature controlled reaction stage must be tested for propertemperature control, the means to determine the start of the enzymaticreaction must be tested for proper sensitivity, the optical means toaccess reaction progress must be tested (light source, light detector,integrity of optical stage, etc.), and finally the computational meansmust be tested. Alternatively partial verification of some of thesubsystems may be done, and the remainder of the subsystems tested byalternate means, such as liquid control solution and a disposable testunit.

To verify the function of such analytical devices, electronicverification or “control” devices or circuits are commonly used. Suchverification devices can simulate the action of an enzymatic sampleinteracting with a disposable. reagent. If the analytical device returnsthe proper answer after analysis of the verification device, then theproper functioning of the analytical device can be verified without theexpense of using the one time use reagent cartridges.

The use of reference paint chips to calibrate and verify photometricdevices has long been known in the art. When applied to home bloodglucose monitors, such reference chips are often referred to as “checkstrip”. For example, the LifeScan One-Touch™ blood glucose monitorincludes a colorimetric “check strip” in with its meter system. This“check strip” consists of an opaque plastic strip with a paint chip ofknown colorimetric properties affixed to it. The check strip is insertedinto the meter, and is used to verify the performance of the meter'scolorimetric photodetector. The system does not vary the intensity ofthe colorimetric paint chip target as a function of time to simulate anormal test reaction, nor does it incorporate means to monitor theanalytical devices' temperature.

Recent refinements to the basic “paint chip” technique, suitable forclinical reagents and instrumentation, include U.S. Pat. Nos. 4,509,959;4,523,852; 4,729,657; 5,151,755; 5,284,770; and 5,592,290. U.S. Pat. No.4,509,959 disclosed an apparatus incorporating many such reference colorchips. U.S. Pat. No. 4,523,852 disclosed a reference standard, suitablefor visually read diagnostic reagent test strips, consisting of manycolored reference areas of differing hues. U.S. Pat. No. 4,729,657disclosed photometer calibration methods using two or more reflectancestandards and using least squares regression line analysis to constructand store calibration curves in the analytical device's memory. U.S.Pat. No. 5,151,755 disclosed methods to detect defects in biochemicalanalysis apparatuses measurement means by irradiating a referencedensity plate with light that has passed through a plurality ofinterference filters and comparing the relative amounts of reflectedlight obtained by these different measurements. U.S. Pat. No. 5,284,770disclosed use of a check strip, along with an analytical instrumenthaving a user insertable key (memory chip) containing the parameters ofacceptable check strip performance, so that correct instrumentperformance can be automatically verified. U.S. Pat. No. 5,592,290disclosed optical analyzer instrument error correction methods usingstandard color plates incorporating dyes with absorption spectrumsimilar to the analytical reagent normally read by the analyzer. Thesestandard color plates are then used in conjunction with a secondreference optical analyzer and a specific correction algorithm tocorrect the instrument error in the first instrument.

In addition to passive “paint chip” verification methods, a number ofdifferent active (typically electronic) verification methods have alsobeen used. These active verification methods typically involveelectronic components, and often produce a dynamic (as opposed to astatic) reference signal to the analytical instrument.

U.S. Pat No. 4,454,752 disclosed a test circuit for use in a photometriccoagulation instrument for plasma samples that verified the electroniccircuitry of the instrument, wherein the rapid rise in clot density of aplasma sample may be simulated by a applying to the clot detectioncircuitry of the instrument a synthetic waveform that simulates thesignal that results during clot formation in a reagent plasma mixture.However, this patent did not disclose methods by which the properfunctioning of an instrument capable of measuring whole blood can beanalyzed. The disclosed methods are capable of verifying only that theclot detection circuits of an photometric plasma coagulation instrumentare performing properly. The patent did not disclose methods by whichother instrument functions such as temperature control, absence ofoptical system light leaks, proper detection of sample insertion, etc.,may also be verified.

Verification methods suitable for partially verifying the function ofcertain whole blood coagulation analyzers and unitized reagentcartridges are also known in the art. For example, U.S. Pat. Nos.4,948,961 and 5,204,525 disclosed a quality control device for aninstrument with an analysis cartridge constructed so that theinstrument's light passes through the cartridge's internal chamber. Suchsystems have been used for a number of whole blood clinical tests,including whole blood prothrombin time assays when the internal chamberis filled with thromboplastin, and the cessation of red cell movement istracked by light scattering techniques.

U.S. Pat. No. 5,204,525 disclosed a control device using a liquidcrystal cell interposed between the light source and detector in ananalytical instrument, and a polarizing filter, so that the passage orblock passage of light between the analytical device's light source andlight detector when the voltage to the liquid crystal is modulated.However, neither U.S. Pat. No. 5,204,525 nor U.S. Pat. No. 4,948,961disclosed means by which the temperature control of an analytical devicemay be verified. Although these publications disclosed devices usefulfor monitoring the function of optically transmissive reaction chambersin which the light source passes through the chamber, and which thereaction in question does not alter the wavelength of the light emittedby the instrument's light source, they did not disclose devices usefulfor monitoring the function of fluorescent test strip articles such asthose disclosed in U.S. Pat. No. 5,418,143. In such systems, light ofone wavelength enters a test strip, and excites a fluorescent compoundwhich then emits light that exits the test strip at the same side as thelight source (rather than passing through a reaction chamber), and at adifferent wavelength.

Another type of control device is found in the Boehringer Mannheim“Coaguchek” whole blood prothrombin time analysis device disclosed byU.S. Pat. No. 4,849,340. This device uses a disposable reagent cartridgeconsisting of a chamber with thromboplastin and magnetic particles. Thedisposable reagent cartridge is placed in a stage in the analysisdevice, and a blood sample is added. The analysis device subjects thereagent cartridge to a varying magnetic field, and detects the motion ofthe magnetic particles by the optical interaction between the motion ofthe magnetic particles and a beam of light. In normal operation, whenblood is applied to the disposable reagent cartridge, the magneticparticles are free to move in suspension, and thus provide a high degreeof modulation to the optical signal in response to the varying magneticfield. As the blood clots in response to the thromboplastin reagent, themagnetic particles become less able to move, and thus provide aprogressively smaller amount of modulation to the optical signal as timeprogresses.

An “electronic control” is provided for the Coaguchek. (BoehringerMannheim electronic control user manual, 1996). This “electroniccontrol” consists of a separate device consisting of a disposablereagent sized probe that fits in to the reagent stage on the Coaguchekdevice. The probe contains a magnetic coil pickup, a light emittingdiode, and means to vary the intensity of the response of the lightemitting diode to current generated by the magnetic coil pickup. Byusing this “electronic control” device, the operator can verify that thevarying magnetic field generator on the Coaguchek is operating properly,and that the optical sensor on the Coaguchek is also operating properly.The temperature of the reaction stage, and the performance of theoptical light source on the Coaguchek, are not tested by this device,however.

In addition to passive (time unvarying reference signal) and active(time varying reference signal) verification devices, a third type ofverification methodology has been disclosed which incorporates certainverification systems on to the disposable reagent itself. This isdisclosed by in U.S. Pat. Nos. 5,591,403 and 5,504,011. U.S. Pat. No.5,591,403 disclosed a reaction chamber cuvette, useful for prothrombintime testing, with multiple conduits. One or more conduits contain thereaction chemistry for the prothrombin time reaction itself, and otherconduits contain control agents useful for assessing certain functionsof the analytical instrument that reads the test cartridge, and the testcartridge itself. Typically one “control” conduit will contain a vitaminK dependent clotting factor concentrate, and a different “control”conduit will contain an anticoagulant. In a properly functioninginstrument, the control conduit with the vitamin K dependent clottingfactor concentrate will initiate a coagulation signal early, and thecontrol conduit with the, anticoagulant will initiate a coagulationsignal late. This tests the proper function of those meter detectorelements that read the status of the control conduits. Because thecontrol elements are incorporated into normal prothrombin time reactioncuvette, an independent, non-destructive, test of proper meter functionis not possible with this system.

Thus, a need exists for an improved verification device. This need andothers are addressed by the instant invention.

SUMMARY OF THE INVENTION

One aspect of the invention is a method for verifying the output of asystem having a radiation source and a radiation detector, said methodcomprising positioning a reference surface to receive radiation from theradiation source and return radiation to the detector; and modulating atleast one of the radiation from the source and the radiation to thedetector over time to emulate reflective or radiation characteristics ofa chemical or biological reaction on the reference surface.

A further aspect of the invention is a method for verifying the outputof a system having a radiation source and a radiation detector, saidmethod comprising positioning a reference surface to receive radiationfrom the radiation source and return radiation to the detector; andmodulating at least one of the radiation from the source and theradiation to the detector over time in response to temperature changes.In some embodiments, the temperature changes are determined within thesystem. In further embodiments, the temperature changes are determinedexternal to the system.

A further aspect of the invention is an apparatus for use in combinationwith an analyzer having a radiation source and a radiation detector,said apparatus comprising a reference surface which produces returnradiation in response to receiving radiation from the source, and meansdisposed adjacent the radiation surface for modulating at least one ofradiation to the reference surface or radiation from the referencesurface. In some embodiments the modulating means modulates theradiation over time to emulate reflective or radiation characteristicsof a chemical or biological reaction on the reference surface. Infurther embodiments the modulation means modulates the radiation inresponse to changes in temperature.

A further aspect of the invention is an electronically controlledoptical reference device useful for the verification of a clinicalanalytical system having an optical detection apparatus, the referencedevice comprising, an opaque optical reference; an optical shutter;means for controlling the percent exposure of the optical reference tothe optical detection apparatus; optionally, a means to monitor thetemperature of a reaction stage of the clinical analytical system; andan algorithm or method that controls the rate at which the opticalreference is selectively revealed to the optical detection apparatus;said algorithm or method being selected as to simulate the reactionrates of one or more levels of clinical analytes reacting with a testreagent.

A further aspect of the invention is a verification device useful fordetermining the proper function of an optical, temperature controlledanalytical instrument, the device comprising an electronic opticalshutter with an optically active backing, interposed between an opticalsignal emitted by the analytical instrument and an optical detectormounted on the analytical instrument; a temperature sensor, the sensorcontacting a reaction stage on the analytical instrument; andverification device electrodes, the verification device electrodesmaking contact with electrodes on the reaction stage of the analyticalinstrument; wherein the action of the device is initiated by aresistance drop across the verification device electrodes, and whereinthe optical transmission of the liquid crystal shutter is modulated as afunction of time and of the temperature of the reaction stage, wherein arange of levels of enzymatic activity measured by the analyticalinstrument at various operating temperatures is simulated.

A further aspect of the invention is a verification device useful fordetermining the proper function of an optical, temperature controlledanalytical instrument, the device comprising an opticalshutter-fluorescent backing assembly comprising an optical shutterhaving a fluorescent backing placed on one side of the optical shutter;the assembly being interposed between an optical signal emitted by theanalytical instrument and an optical detector mounted on the analyticalinstrument; a thermocouple in contact with a reaction stage on theanalytical instrument; and verification device electrodes, theverification device electrodes making contact with electrodes on thereaction stage of the analytical instrument; wherein the action of thedevice is initiated by a resistance drop across the device electrode,and wherein the fluorescence of the optical shutter-fluorescent backingassembly is modulated as a function of time and of the temperature ofthe reaction stage, wherein a range of levels of enzymatic activitymeasured by the analytical instrument at various operating temperaturesis simulated.

A further aspect of the invention is an electronically controlledoptical reference device, useful for the verification of an analyticalinstrument having an optical detection apparatus and using opticallyread reagent test strips, the device comprising an opaque opticalreference, which simulates the optical characteristics of a reagent teststrip after reaction with its intended clinical sample; an opticalshutter; a means for controlling the percent exposure of the opticalreference to the optical detection apparatus; and an algorithm or methodthat controls the rate at which the check strip is selectively revealedto the optical detection apparatus, said algorithm or method beingselected as to mimic the reaction rates of one or more levels ofclinical analytes reacting with a reagent test strip.

A further aspect of the invention is a method for verifying the correctperformance of a clinical analytical system comprising an opticaldetection apparatus, the method comprising contacting the clinicalanalytical system with an electronically controlled optical referencedevice useful for the verification of clinical devices using opticallyread reagent test strips, the reference device comprising, an opaqueoptical reference, which simulates the optical characteristics of areagent test strip after reaction with its intended clinical sample; anoptical shutter; means for controlling the percent exposure of theoptical reference to the optical detection apparatus; optionally, ameans to monitor the temperature of the clinical analytical system; andan algorithm or method that controls the rate at which the opticalreference is selectively revealed to the optical device, said algorithmor method being selected so as to mimic the reaction rates of one ormore levels of clinical analytes reacting with a reagent test strip; andanalyzing the optical reference; wherein an expected result of analysisof the optical reference by the clinical analytical system is predictiveof the correct performance of the clinical analytical system.

A further aspect of the invention is a method for verifying thetemperature control of a clinical analytical system comprising anoptical detection apparatus, the method comprising contacting theclinical analytical system with a verification device useful fordetermining the proper function of an optical, temperature controlledanalytical instrument, the device comprising an electronic opticalshutter with an optically active backing, interposed between an opticalsignal emitted by the analytical instrument and an optical detectormounted on the enzymatic analytical instrument; a temperature sensor,the sensor contacting a reaction chamber on the analytical instrument;and verification device electrodes, the verification device electrodesmaking contact with electrodes on the reaction chamber of the analyticalinstrument; wherein the action of the device is initiated by aresistance drop across the verification device electrodes, and whereinthe optical transmission of the liquid crystal shutter is modulated as afunction of time and of the temperature of the reaction chamber, whereina range of levels of enzymatic activity measured by the analyticalinstrument at a range of operating temperatures is simulated, andanalyzing the optical reference; wherein an expected result of analysisof the optical reference by the clinical analytical system is predictiveof the correct operating temperature of the reaction chamber of theclinical analytical system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting an electronic strip, emulator constructedaccording to the principles of this disclosure (outside the dotted box),interacting with an exemplary test device (inside the dotted box).

FIGS. 2A and 2B depict two types of optical shutters. The shutter inFIG. 2A comprises a single shutter element, which can be graduallyvaried from non-transmissive is to transmissive. The optical shutter inFIG. 2B comprises numerous shutter “pixel” elements.

FIG. 3 is a graph depicting the output from the electronic verificationdevice when the analytical device (meter) is at a normal temperature(37° C.), and at an aberrant temperature (33° C.). Here, Level I mimicsa prothrombin time reaction curve obtained from a test sample with anormal prothrombin time value, and Level II mimics the prothrombin timereaction curve obtained from a test sample with an elevated prothrombintime value.

FIG. 4 depicts an example of a temperature correction algorithm alteringthe kinetics of fluorescence development in response to the timeaveraged temperature readings from the electronic verification device.The temperature algorithm is selected to match the temperature responseof a real reagent test strip.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a method for verifying the output of asystem having a radiation source and a radiation detector, said methodcomprising positioning a reference surface to receive radiation from theradiation source and return radiation to the detector; and modulating atleast one of the radiation from the source and the radiation to thedetector over time to emulate reflective or radiation characteristics ofa chemical or biological reaction on the reference surface. In someaspects of the invention, the modulator may operate without thereference surface by selectively reflecting light.

The present invention also provides a method for verifying the output ofa system having a radiation source and a radiation detector, said methodcomprising positioning a reference surface to receive radiation from theradiation source and return radiation to the detector; and modulating atleast one of the radiation from the source and the radiation to thedetector over time in response to temperature changes. In someembodiments, the temperature changes are determined within the system.In further embodiments, the temperature changes are determined externalto the system.

The present invention also provides an apparatus for use in combinationwith an analyzer having a radiation source and a radiation detector,said apparatus comprising a reference surface which produces returnradiation in response to receiving radiation from the source, and meansdisposed adjacent the radiation surface for modulating at least one ofradiation to the reference surface or radiation from the referencesurface. In some embodiments the modulating means modulates theradiation over time to emulate reflective or radiation characteristicsof a chemical or biological reaction on the reference surface. Infurther embodiments the modulation means modulates the radiation inresponse to changes in temperature.

The present invention also provides a verification device for a clinicalanalytical system or instrument. Such a device is also referred toherein as a test strip emulator, a control test simulator, and anelectronically controlled optical reference device. The verificationdevice is typically an electronically controlled optical referencedevice useful for the verification of a clinical analytical systemhaving an optical detection apparatus. The reference device comprised anopaque optical reference or “target”, an optical shutter, and means forcontrolling the percent exposure of the optical reference to the opticaldetection apparatus. The opaque optical reference preferably simulates acolorimetric, fluorescent, or luminescent reagent test strip.Optionally, a means to monitor the temperature of a reaction stage ofthe clinical analytical system is included as part of the device. Thedevice is preferably programed with an algorithm or comprises a methodthat controls the rate at which the optical reference is selectivelyrevealed to the optical detection apparatus. Typically the selectiverevealing of the optical reference is done by exposing the opticalreference to the optical detection apparatus over a specified timeinterval. The algorithm or method is selected so as to simulate thereaction rates of one or more levels of clinical analytes reacting witha test reagent. The algorithm or method can be modified to account forthe temperature of a reaction stage or chamber in the analytical system.

In some embodiments of the invention, the device of comprises one ormore first electrodes, wherein electrodes contact one or more secondelectrodes on a reaction stage of the clinical analytical system. Theelectrical resistance across the electrodes in the reference device ismodulated to simulate the addition or removal of a disposable reagenttest strip or cartridge or a liquid sample to the clinical analyticalsystem.

The optical shutter of the device may be electronically operated.Exemplary shutters include but are not limited to a liquid crystalshutter, a magneto-optical shutter, a Faraday effect optical shutter, athermochromic optical shutter, an electrochromic optical shutter, or amicro-mechanical optical shutter. The optical shutter may be dividedinto a plurality of independently or semi-independently controlled pixelelements, such that the optical shutter modulates the intensity of anoptical signal by varying the optical state of the shutter pixels in atime dependent manner.

In some embodiments, the optical shutter comprises a fluorescent backingon one side of the optical shutter, and a first optical signal of afirst wavelength passes through the optical shutter and interacts withthe fluorescence backing, and a fluorescence signal of a secondwavelength passes back through the optical shutter. In furtherembodiments, the optical shutter comprises a colored backing on one sideof the optical shutter, and a first optical signal consisting of a firstspectrum of wavelengths passes through the optical shutter and interactswith the colored backing, and a second optical signal consisting of asubset of the first spectrum of wavelengths passes back through theoptical shutter. The optical shutter may also comprise a luminescentbacking on one side of the optical shutter, with the optical signalcomprising a time increasing or time decreasing luminescence signal.

In a further feature of the invention, a thermocouple monitors thetemperature of the reaction stage of the analytical device. Thetransparency of the optical shutter may be modulated as a function oftime and of a thermocouple monitored temperature of the reaction stage,wherein a range of levels of enzymatic activity measured by theanalytical system at various operating temperatures is simulated.

In some embodiments of the invention, the verification device provides ameans to monitor a reagent present and/or blood present sensor on theclinical analytical device, wherein a stimulus to these sensors isprovided to signal readiness of the meter for testing a clinical sample.

The instant invention also provides a verification device useful fordetermining the proper function of an optical, temperature controlledanalytical instrument. Typically such a device will comprise anelectronic optical shutter with an optically active backing, interposedbetween an optical signal emitted by the analytical instrument and anoptical detector mounted on the analytical instrument; a temperaturesensor, the sensor contacting a reaction stage on the analyticalinstrument; and verification device electrodes, the verification deviceelectrodes making contact with electrodes on the reaction stage of theanalytical instrument. The action of the device is preferably initiatedby a resistance drop across the verification device electrodes. Theoptical transmission of the liquid crystal shutter is modulated as afunction of time and of the temperature of the reaction stage, wherein arange of levels of enzymatic activity measured by the analyticalinstrument at various operating temperatures is simulated. The reactionstage of the analytical device may be heated.

In some embodiments the device comprises an optical shutter-fluorescentbacking assembly comprising an optical shutter having a fluorescentbacking placed on one side of the optical shutter; the assembly beinginterposed between an optical signal emitted by the analyticalinstrument and an optical detector mounted on the analytical instrument;a thermocouple in contact with a reaction stage on the analyticalinstrument; and verification device electrodes, the verification deviceelectrodes making contact with electrodes on the reaction stage of theanalytical instrument. The action of the device is initiated by aresistance drop across the device electrode. The fluorescence of theoptical shutter-fluorescent backing assembly is modulated as a functionof time and of the temperature of the reaction stage, wherein a range oflevels of enzymatic activity measured by the analytical instrument atvarious operating temperatures is simulated. The reaction stage of theanalytical device may be heated.

The instant invention also provides an electronically controlled opticalreference device, useful for the verification of an analyticalinstrument having an optical detection apparatus and using opticallyread reagent test strips. The device comprises an opaque opticalreference, which simulates the optical characteristics of a reagent teststrip after reaction with its intended clinical sample; an opticalshutter; a means for controlling the percent exposure of the opticalreference to the optical detection apparatus, and an algorithm or methodthat controls the rate at which the check strip is selectively revealedto the optical detection apparatus, said algorithm or method beingselected as to mimic the reaction rates of one or more levels ofclinical analytes reacting with a reagent test strip. In a preferredembodiment, the reagent is thromboplastin.

The instant invention also provides a method for verifying the correctperformance of a clinical analytical system comprising an opticaldetection apparatus using the devices of the instant invention. In apreferred embodiment, the clinical analytical system is contacted withan electronically controlled optical reference device useful for theverification of clinical devices using optically read reagent teststrips. The reference device, for example, comprises an opaque opticalreference, which simulates the optical characteristics of a reagent teststrip after reaction with its intended clinical sample; an opticalshutter; means for controlling the percent exposure of the opticalreference to the optical detection apparatus; optionally, a means tomonitor the temperature of the clinical analytical system; and analgorithm or method that controls the rate at which the opticalreference is selectively revealed to the optical device, said algorithmor method being selected as to mimic the reaction rates of one or morelevels of clinical analytes reacting with a reagent test strip. Anexpected result of analysis of the optical reference by the clinicalanalytical system is predictive of the correct performance of theclinical analytical system.

The instant invention also provides a method for verifying thetemperature control of a clinical analytical system comprising anoptical detection apparatus using the devices of the instant invention.In a preferred embodiment, the method comprises contacting the clinicalanalytical system with a verification device useful for determining theproper function of an optical, temperature controlled analyticalinstrument. For example, the device comprises an electronic opticalshutter with an optically active backing, interposed between an opticalsignal emitted by the analytical instrument and an optical detectormounted on the enzymatic analytical instrument; a temperature sensor,the sensor contacting a reaction chamber on the analytical instrument;and verification device electrodes, the verification device electrodesmaking contact with electrodes on the reaction chamber of the analyticalinstrument. The action of the device is initiated by a resistance dropacross the verification device electrodes, and wherein the opticaltransmission of the liquid crystal shutter is modulated as a function oftime and of the temperature of the reaction chamber, wherein a range oflevels of enzymatic activity measured by the analytical instrument at arange of operating temperatures is simulated. An expected result ofanalysis of the optical reference by the clinical analytical system ispredictive of the correct operating temperature of the reaction chamberof the clinical analytical system.

The verification device of the invention can be provided as a probe,suitable for insertion into the reaction chamber of a colorimetric,fluorescent, or chemiluminescent test analytical instrument, with anopaque colorimetric, fluorescent or luminescent target. The reflectance,fluorescence or luminescence of the target is modulated by an opticalshutter. Typically the probe will additionally contain a temperaturesensor, a clock, and means to modulate the optical exposure of theprobes target area according to a preset algorithm. The preset algorithmis designed to mimic the response of a normal reagent with one or morelevels of test analyte. The probe may optionally contain other elementsdesigned to interact with and test other meter functional elements, suchas a meters “reagent present” and “sample present” sensors.

The optical characteristics of the opaque optical target can varydepending upon the analytical device in question. In one embodiment, thetarget is optically reflective and/or optically colored, so as toeffectively change the distribution of various wavelengths or lightintensity of the target as a function of the state of the opticalshutter. In a further, embodiment, the target can made of a fluorescentmaterial, so that the fluorescent intensity of the light detected by theanalytical system's detector varies as a function of the state of theoptical shutter. In a third embodiment, the backing can be luminescent(for example, an electronic luminescent panel), so that the luminescenceseen by the analytical system's luminescence detector varies as afunction of the state of the optical shutter. Although for brevity, thisdiscussion will focus on fluorescent targets, it should be understoodthat the same principles would also apply to colorimetric or luminescentanalytical systems as well.

A fluorescent target is typically composed of a fluorescent compound,with absorption and emission characteristics similar to that of theanalytical devices normal fluorescent reagent. The compound willtypically be incorporated into a rigid support matrix. This can be doneby mixing the fluorescent target compound with a suitable supportcarrier, such as acrylic paint, epoxy, or the like. To maximize theoptical signal-to-noise characteristics of the fluorescent target,sufficient quantities of fluorescent compound are added as to completelyinteract with the entire fluorescence optical excitation signal,rendering the target optically opaque. The fluorescent target shifts thewavelength of the excitation signal to a different wavelength, and thefluorescent signal emerges from the side of the target that isilluminated by the excitation wavelength.

Alternatively, if it is infeasible to make the target opaque using largeamounts of fluorescent compound, the back of the target may painted withan opaque backing. The characteristics of this opaque backing may beselected to maximize the signal-to-noise performance of the fluorescenttarget. If the optical cutoff efficiency of the fluorescent detector'sfilters to the fluorescence excitation wavelengths is high, the opaquebacking could be selected to be of a shiny reflective material.Alternatively, if the optical cutoff efficiency of the fluorescentdetector's filters to the fluorescence excitation wavelengths is lower,a non-reflective (black) opaque backing may be chosen to minimize backreflections of the incoming excitation wavelengths to the fluorescencedetector.

In yet another embodiment, the target may be luminescent, and used in achemiluminescence detecting analytical device that has an opticaldetector, but does not contain a light source. The light source for theluminescent target may be provided by variety of conventional electricallighting techniques.

The optical shutter may be a mechanical or electromechanical shutter,such as an iris as typically used to control exposure intensity incameras, a series of louvers, or the like. Alternatively, the opticalshutter may be an electro-optical shutter, such as a liquid crystalshutter, a magneto-optical electric shutter, Faraday effect opticalshutter, thermochromic optical shutter, Electrochromic optical shutter,micro-mechanical optical shutter, or other such device. Some exemplaryoptical stutters suitable for the present invention are disclosed, forexample, in U.S. Pat. Nos. 3,649,105; 4,556,289; 4,805,996; 4,818,080;5,050,968; 5,455,083; 5,459,602; and 5,525,430.

The optical shutter may be composed of a single functional shutterelement, or alternatively it may be composed of many smaller functionalshutter elements, that collectively act to act to alter the opticalcharacteristics of the shutter as a whole.

In an configuration, the shutter is mounted so that light illuminatingthe optical (fluorescent) target passes through the shutter. Fluorescentlight re-emitted by the optical target may pass directly to theanalytical device fluorescence detector, or optionally pass through theoptical shutter on the way to the fluorescence detector. Alternatively,the optical shutter can be mounted to interact only with light emittedby the optical target. In still a further configuration, the opticalshutter can interact with light both illuminating and emitted by theoptical target.

The device may optionally contain means of monitoring the temperature ofthe probe near the target area, as well as means of modulating thefluorescence signal in response to the temperature of the target area.These means may be mechanical, such as a bimetallic strip mechanicaltemperature sensor device hooked up to a mechanical shutter, chemical,such as a temperature sensitive liquid crystal thermometer, orelectronic, such as a thermistor, thermocouple, or the like. In thepreferred embodiment, the temperature sensor is electronic.

The means to modulate the target's fluorescence may be mechanical, suchas a clockwork mechanism, cam, or the like. The means may be controlledby analog electrical circuits, such as simple analog timers or the like,or the means may be controlled by digital electrical circuits, such asmicroprocessors, microcontrollers, and the like. In the case ofmechanical means, the algorithm encoding the state of the target'sfluorescence as a function of time is encoded into the design of themechanical timing elements. In the case of analog electrical circuits,the algorithm is encoded by properly selected time constants, and thelike. In the preferred case of digital microprocessor controllers, thealgorithm is encoded by a specific program that controls fluorescence asa function of time, and optionally temperature and other variables.

To fully validate the analytical system's performance over a variety ofsample ranges, the algorithm will ideally simulate the reactionoccurring when samples with different relative activity react with testreagents. The algorithm may switch from simulating one test level to adifferent test level either in response to user input, or automaticallyas the test algorithm runs through a preset series of validation tests.

The verification device's probe may optionally contain one or moreadditional elements that interact with and validate other aspects of theproper function of the analytical system. For example, the probe maytest the function of systems that determine if a cartridge has beenproperly inserted, or systems that determine if sufficient sample hasbeen added. In the case of optical strip insertion or sample additionschemes, the probe may contain additional light emitting or lightblocking devices designed to interact with appropriate optical detectorson the instrument. Alternatively, in the case of electronic stripinsertion or sample addition schemes, such as those disclosed in U.S.Pat. Nos. 5,344,754 and 5,554,531, and in Zweig et. al., BiomedicalInstrumentation & Technology 30: 245–256 (1996), the probe may containone or more electrodes that interact with corresponding electrodesensors on the analytical device, and provide appropriate inputs tosimulate normal activity.

The verification device may be constructed as a stand-alone,independently powered unit. This may be manually inserted or removed bythe user, or inserted or removed by automated equipment. Alternatively,the verification device may be constructed as an integral part of theanalytical device itself, and may share one or more elements (powersupply, microprocessor time, memory, etc.) with the analytical device.

A schematic diagram of the verification device of the instant inventioninteracting with a meter is depicted in FIG. 1. In this embodiment, themeter 40 consists of an electrically heated support stage 27, containingan optical window 30 through which light 22 emitted from light source 21can pass. The meter additionally contains an optional fluorescencefilter 24 and a photodetector 26. In normal use, light 22 travelsthrough the optics window 30 and illuminates a fluorescent reagenttarget. Fluorescent light 23 travels though filter 24 and afterfiltration illuminates photodetector 26. The meter is controlled by amicroprocessor 29, which initiates test timing in response to inputsfrom strip detect and sample detection electrodes 28. The verificationdevice circuit board additionally contains electrodes 14 that interactswith the strip detect and blood detect electrodes 28 on the meter'soptics block 20. A thermocouple 11 performs an independent measurementof the temperature of the meter's heated optics block 27. Theverification device has an optical shutter 12, with a backing 13, and acircuit board 10. A microcontroller 15 is also provided.

In a preferred embodiment, the verification device has an 8×8 pixelliquid crystal optical shutter 12, with an active area of 0.375×0.375″,and an exterior size of 0.5″×0.6″, made by Polytronics, Inc. This isplaced on a 0.02″ thick circuit board 10, with exterior dimensions of0.75″, and length of 2″. The exterior circuit board is made to the samesize as a disposable test strip unit normally used in an Avocet Medicalprothrombin time detector (see Zweig, et.al., Biomedical Instrumentation& Technoloay 30, 245–256; FIGS. 3 and 4).

The optical shutter has a backing 13 consisting of Rhodamine 110 mixedwith epoxy. The rhodamine 110 retains its normal fluorescence activitywhen mixed with the epoxy, and the epoxy provided a way to affix theRhodamine 110 to the back of the optical shutter 12 in durable andpermanent manner. The active elements on the circuit board arecontrolled by a Texas Instruments TSS400-S3 sensor signal processor 15,which is a combination microcontroller, liquid crystal display driver,and A/D converter. The TSS400 additionally contains 2K bytes ofprogrammable EEPROM, which contained the algorithm needed to drive thesystem. When turned on (switches not shown), the verification deviceinitially turns all 64 pixels of the 8×8 pixel optical shutter to theopaque mode. Electrodes 14 connecting to the strip present sensors 28 onthe meter's optics block 20 are switched to conducting mode (resistanceis lowered), to allow the Avocet Meter to detect that a test strip isinserted into the optics block. The meter then initiates a warm-upsequence.

In this preferred embodiment, upon reaching proper temperature, themeter then sends a signal via its sensor electrodes 28 to theverification device electrodes 14 informing the verification device thatthe meter is now warmed up. Alternatively, the meter can signal to theuser that it is ready, and the user can manually transfer thisinformation to the verification device by pressing an electrical switchon the verification device. After the verification device is informedthat the meter is now ready to proceed, the device then reduces theresistance across a second set of electrodes 44, which interact with theblood present sensors 42 on the meter's optics block. Whereas thisresistance drop is normally used to signal the application of blood tothe reagent strip (see, for example, U.S. Pat. No. 5,344,754), in theinstant invention it is used to signal the meter to proceed even thoughno blood has actually been applied.

In this preferred embodiment, the microcontroller 15 on the verificationdevice consults an algorithm, and selectively switches an increasinglylarger number of pixels on the liquid crystal shutter 12 to transparentmode as a function of a number of variables, including time, the settingof the verification device (e.g. Level I or II control, etc.), andoptionally the temperature of the meter's optics stage 27 as measured bytemperature sensor 11. The meter optical system 20 observes thefluorescent backing 13 through the optical shutter 12, and observes aprogressive increase in overall fluorescence as a function of time.Alternatively, the verification device can progressively alter thevoltage applied to a single element optical shutter element (see FIG.2A) so as to progressively increase the transparency of the singleelement shutter as a function of time and temperature.

Exemplary algorithms are as follows. The verification device containsone or more stored reaction profile algorithms that control the percentlight transmission of the optical shutter as a function of time. Apreferred Level I algorithm is:% fluorescence (% pixels switched on)=100*[0.01*Reactiontime−.0.35]  Equation 1:% fluorescence (% pixels switched on)=100*[0.02*Reactiontime−0.9]  Equation 2:% fluorescence (% pixels switched on)=100*[0.01*Reactiontime−0.05]  Equation 3:

In a preferred embodiment every ten seconds (the frequency at which themeter took data) for 240 seconds (a typical test duration) theverification device computes all three equations, and chose the resultsbased upon the rule:

-   If Equation 1<0, % fluorescence=0;-   If Equation 1>0 and <20%, % fluorescence=Equation 1-   If Equation 1>20% and Equation 2<80%, % fluorescence=Equation 2-   If Equation 2>80% and Equation 3<100%, % fluorescence=Equation 3-   If Equation 3>100%, % fluorescence=100%.

This produces an “S” shaped reaction profile, shown in FIG. 3, similarto that of a normal prothrombin time (Level I) sample. In contrast, anexemplary algorithm used for the elevated prothrombin time (Level II)control is:Equation 1, 2, and 3: % fluorescence (% pixels on)=100*[0.007*Reactiontime−0.6]

The decision tree is the same as the Level I control shown previously.This produces a delayed, lower slope, linear curve more typical of thatof a normal Level II sample reaction.

A typical temperature verification algorithm is as follows. To verifythat the meter's optical stage is being maintained at the propertemperature, the thermocouple on the electronic verification deviceperiodically (every second) performs a temperature measurement. Theresults from each temperature are converted into degrees C. ifnecessary. The degree of deviation of the measured temperature from theideal temperature is used as input into a temperature correctionalgorithm. This temperature correction algorithm advances or retards theschedule of pixel switching on the optical shutter in such a way as tomimic the response of a normal test strip-reagent reaction reacting at adeviant temperature. For a prothrombin time reaction, previous work(Daka et al., Journal of Investigative Surgery 4: 279–290; 1991), hasshown that the optimal reaction temperature for a prothrombin time testis at a reaction time minima, and deviations from this idealtemperature, either positive or negative, prolong the prothrombin timevalue.

In a normal reagent reaction, the effects of temperature are cumulative.That is, a reaction held at the proper temperature for 95% of thereaction will be only mildly affected if the temperature is slightlydeviant during 5% of the reaction. For an exemplary prothrombin timereaction, the effects of non-ideal temperature (either positive ornegative) is to slow down the reaction (the ideal temperature is at areaction time minima). Our own work, as well as the work of Daka et.al., has shown that for the prothrombin time verification devicediscussed by example here, the effects of non-ideal temperatures on theprothrombin time reaction can be approximated by the equation:Fluorescence(Time,Temp)=Fluorescence(Time−(a*(Temperature Deviation)2),where “a” is an experimentally determined coefficient (here taken to be0.1) used to bring the validation device's temperature variation in linewith those of an actual reagent test strip.

The verification device reaction profile, described by the Level I andII equations above, can be temperature compensated to match a normalreagent reaction profile, reacting at a deviant temperature, bysubtracting a factor proportional to the time weighted temperaturedeviation average from the % fluorescence calculation at each relevanttime point. This delays the onset of fluorescent development. Higherorder polynomial fits, or other temperature compensation functions, canalso be used for these purposes.

The meter's microcontroller takes a series of fluorescence readings asfor a normal test strip, and interprets the result according to itsnormal test strip algorithm., Previous work (U.S. Pat. No. 4,418,141 andthe Zweig et. al., supra), has shown that for the prothrombin timeexample illustrated here, the prothrombin time (PT) time correlateslinearly with the time at which the normalized fluorescence reactionprofile first exceeds 10% of its maximum value (Time 10%). Thus byshifting the time at which the verification device reaction profilefirst exceeds 10% of its maximum value; the temperature compensationalgorithm will cause a corresponding shift in the prothrombin time valuereported by the meter after reading the verification device.

FIG. 3 depicts the verification devices' fluorescence profiles reactingaccording to the Level I and Level II algorithm at a simulated optimaltemperature of 37° C., and at an aberrant lower temperature. FIG. 4shows graphically how the verification device's temperature correctionalgorithm delays the initial onset of the fluorescent signal (Time 10%)for meters operating at aberrant temperatures.

If the meter is working properly, the answer displayed will be withinthe expected parameters. If the is fluorescence detector is workingimproperly, the meter's internal error detection mechanisms will detecta problem (no signal or erratic signal) and display an error code. Ifthe meter's stage is at the incorrect temperature, the deviant patternof pixel switching on the electronic strip emulator causes the meter tooutput an answer outside of the expected parameters. The user can eitherbe instructed to not use the system when this happens, and or the metercan itself examine the results, and automatically lock itself into a“safe” mode to prevent outputting an erroneous answer when a real sampleis used.

EXPERIMENTAL EXAMPLES

The device constructed according to the preferred embodiment disclosedabove was constructed. A fluorescent backing was provided by firstmixing 50 mg/ml of Rhodamine-123 in 10 ml of isopropyl alcohol solution,to produce a 5 mg/ml Rhodamine-123 solution. This was mixed in a 1:3ratio with “Clear Gloss” acrylic finish (lot 404100, Floquil-Pollt SColor Corp., Amsterdam N.Y.) The Rhodamine dye mixed evenly with thisacrylic, paint. 10 microliters of this acrylic paint-dye mix was thenapplied to the back of a Polytronics liquid crystal shutter, and allowedto air dry. When dry, the paint formed a clear durable finishencapsulating the Rhodamine-dye. After drying, the back of the liquidcrystal shutter was further coated with black epoxy, forming a moredurable, and light opaque fluorescence backing.

The verification device was then programmed as described above for thepreferred embodiment, and tested in a prototype Avocet PT-1000prothrombin time instrument. The instrument responded to theverification device as it would to a normal test strip reacting withsample, and produced appropriate Level I (normal prothrombin time) andappropriate Level II (prolonged prothrombin time) answers.

All references (including appendices, books, articles, papers, patents,and patent applications) cited herein are hereby expressly incorporatedby reference in their entirety for all purposes.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodification, and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice in the artto which the invention pertains and as may be applied to the essentialfeatures hereinbefore set forth, and as fall within the scope of theinvention and the limits of the appended claims.

1. A reference device useful for verification of a clinical analyticalsystem having an optical detection apparatus, the reference devicecomprising: an optical shutter; an opaque optical reference comprising afluorescent target with a fluorescent compound incorporated into asupport matrix so that any fluorescent intensity of light detected bythe system varies as a function of the state of the optical shutter;means for controlling the percent exposure of the optical reference tothe optical detection apparatus; optionally, a means to monitor thetemperature of a reaction stage of the clinical analytical system; andan algorithm or method that controls the rate at which the opticalreference is selectively revealed to the optical detection apparatus,said algorithm or method being selected as to simulate the reactionrates of one or more levels of clinical analytes reacting with a testreagent.
 2. The reference device of claim 1, wherein the optical shuttercomprises a fluorescent backing on one side of the optical shutter. 3.The reference device of claim 1, wherein the opaque optical referencecomprises a fluorescent or luminescent reagent test strip.
 4. Thereference device of claim 1, wherein the device provides a means tomonitor a reagent present sensor and/or a blood present sensor, whereina stimulus to the sensors is provided to signal readiness of a meter fortesting a clinical sample.
 5. The reference device of claim 1, whereinthe clinical analytical system is an assay system.
 6. The referencedevice of claim 1, wherein the reference device is selected from thegroup consisting of test strip emulator, a control test simulator, andan electronically controlled optical reference device.
 7. The referencedevice of claim 1, wherein the clinical analytes are enzymatic.